I. Field of the Invention
This invention relates to novel pro-drugs and methods for targeting and regulating the delivery of the pro-drugs to desired cells and tissues. More particularly, the invention relates to the use of transferred genes, preferably reporter or marker genes, with matched pro-drugs that are activated only in the transformed cells and the use of such systems for selecting transformed cells, affecting the cell growth or characteristics of the transformed cells, or as a means of drug targeting to specific cells or tissues in a mixed cell population.
II. Description of the Prior Art
Targeted and Regulated Drug Delivery.
Targeted and regulated delivery of biochemical agents has been investigated for a variety of uses. For example, targeted drug delivery systems have the potential to provide a mechanism for delivering cytotoxic agents directly to cancerous cells, tissue specific drugs to the lung epithelium for cystic fibrosis treatment, or analgesic drugs for chronic (neuropathic) pain treatment. In cancer treatment, the selective delivery of cytotoxic agents to tumor cells is desirable because systemic administration of these agents often kills normal cells within the body as well as the tumor cells sought to be eliminated. Antitumor drug delivery systems currently in use typically utilize a cytotoxic agent conjugated to a tumor-specific antibody to form an immunoconjugate. This immunoconjugate binds to tumor cells and thereby “delivers” the cytotoxic agent to the site of the tumor. Despite the amount of research directed toward the use of immunoconjugates for therapeutic purposes, several limitations involved in these delivery approaches have become apparent (see, e.g., Embleton, Biochem. Society Transactions, 14: 393, 615th Meeting, Belfast, 1986). For example, the large amount of drug required to be delivered to the target tumor cell to effect killing of the cell is often unattainable because of limitations imposed by the number of tumor-associated antigens on the surface of the cells and the number of drug molecules that can be attached to any given antibody molecule.
Methods of cloning genes for bioactive compounds directly into the cells to be affected also have been attempted. The use of cloned gene expression for cell regulatory purposes typically involves effecting cell ablation using a gene expression event. Attempts to express a toxic gene product in cells have not been generally successful. Toxic gene products are difficult to regulate. In addition, cells expressing a toxic product often down-regulate the expression of this toxic product in order to survive. Selection for cells that can survive despite the cloned gene activity also occurs. For example, the diphtheria toxin A-subunit has been transcribed under regulation of a developmentally regulated promoter system in tobacco plants for specific tissue ablation (Koltunow et al., 1990). Mariani et al. (1990) described expression of a chimeric ribonuclease gene to destroy reproductive cells in plants. Recent reports have described selective release strategies for naturally occurring, inactive phytohormone glycoside conjugates (β-glucosides) in cloned plant tissues using a Zea mays cDNA in tobacco roots (Brzobohaty, B., et al, 1993). Gene therapy techniques that include use of a chimeric gene encoding a fusion protein capable of increasing activity of pyrimidine analogs (Tiraby, R., et a., 1996) or combined suicide-gene techniques (Chen, S. H., et al., 1996) have also been reported.
Marker Genes.
Studies in the genetic and molecular basis of eukaryotic growth and differentiation have led to advances in many important areas, including the control of the cell cycle during development, the mechanism of the maternal effect on embryogenesis, and the molecular genetic basis of pattern formation. A byproduct of these studies has been the development of “reporter” or “marker” genes that are used as tools in such work. Studies with a variety of organisms and experimental systems have revealed that many important aspects of eukaryotic development are controlled by the differential expression of genetic information. Tissue specific promoters involved in the expression of developmentally important genes have been identified in various species. Marker genes are commonly used to monitor the effectiveness of these promoters.
Marker genes have been used outside of the study of tissue specific promoters. Expression of foreign genes in mammalian and plant cells has become the quintessential biotechnology protocol, and co-expressed marker genes have been used for several decades to track the expression levels of the simultaneous cloned genes (Roederer, M., et al., 1991). The activity encoded by a chimeric gene construct introduced by techniques of genetic engineering is sensitive, even to the point of measuring activity in single cells (Naleway, 1992; Naleway, et al., 1991). It has become conventional to construct and study gene fusions in which marker gene activity is restricted to particular cell types, tissues, organs, or stages of development.
Marker genes also have been used in combination with a suitable substrate to provide for detection of the protein expression. The substrate is applied to transgenic cells containing an active reporter or marker gene coding for an enzyme, the substrate is enzymatically turned over to a product that can be easily detected by visual or spectrophotometric techniques. Examples of such systems include the use of chromogenic substrates (like 5-bromo-4-chloroindolyl galactoside (X-Gal) for detection of lacZ -galactosidase activity in cells and tissues (Lim, K., et al., 1989; Marsh, J., 1994)), fluorogenic substrates (like 4-methylumbelliferyl glucuronide (MUG) for detection of -glucuronidase activity in plant cells or tissues (Jefferson, R. A., 1988)), or bioluminescent substrates (like luciferin for detection of cloned firefly luciferase activity in various cell or tissue types (Wood, K. V., et al., 1989)). In a related technique, a selection marker (gene) may also be used to confer antibiotic resistance to cells, tissues or organisms when a matched antibiotic is applied to the transgenic cells.
Certain qualities are necessary in a useful marker gene: typically, the lack of detectable intrinsic enzyme activity in the recombinant cells, the robust nature of the marker (usually a bacterial enzyme) and the availability of substrates to estimate the enzyme cloned activity allow sensitive detection of the marker gene (and therefore of any co-expressed gene of interest). The marker genes that are available for use during the genetic engineering of plants or animal cells have found widespread use in molecular biology and biotechnology for the selection, detection and analysis of transgenic cells or tissues.
E. coli lacZ Marker Gene.
The most frequently used reporter gene is probably the Escherichia coli lacZ gene, which encodes an active subunit of β-galactosidase (Lis, et al., 1984 and Beckwith, et al., 1970). The bacterial lacZ 3-Galactosidase enzymatic activity can be easily and sensitively measured, it can be expressed and assayed in virtually any type of cell, and its activity is unaltered by making N-terminal fusion polypeptides.
Firefly luciferase (luc) Marker Gene.
The luc gene from the American firefly Photinus pyralis (de Wet, et al., 1985, de Wet, et al., 1987, Brasier and Ron, 1992) has been widely used as a reporter of cloned gene activities in both plant and animal cells and tissues, both in vivo and in vitro using an assay with added ATP and appropriate buffer (containing Mg+2). This assay is extremely sensitive, allowing detection of subattomole concentrations of enzyme.
Amp Selection Marker.
The selection markers for antibiotic resistance (i.e. amp for ampicillin resistance and tet for tetracycline resistance) are also some of the most widely used marker genes, now routinely incorporated into bacterial plasmid vectors (Bolivar, et al., 1977a; Bolivar, et al., 1977b). Since the (second) tetracycline resistance gene of many plasmids (e.g. pBR322) often contains the cloning site, this leaves ampicillin resistance as a major method of screening recombinant cells in many industrial biotechnology systems. Recombinant cells become capable of detoxifying the antibiotic ampicillin (applied to the media at a concentration of about 50 μg/mL), and are “selected”, while non-transformed cells are ablated.
Other Marker Genes.
A list of common marker genes with their detection methods is given below:
Reporter geneDetection method (reagent)Acid phosphataseColorimetricAequorin (phot)Bioluminescent (coelenterazine)ColorimetricAlcohol dehydrogenaseColorimetricAlkaline phosphataseBL (luciferin phosphate)Chemiluminescent (CSPD)Colorimetric (PNPP)Colorimetric (BCIP)Colorimetric (AS-MXP)Aminoglycoside phosphotransferase (aphAutoradiography ([14C]chloramphenicol)(3′) II) Catechol 2,3-dioxygenase (xylE)Fluorescence (Bodipy chloramphenicol)Chloramphenicolacetyltransferase (CAT)Scintillation Counting ([3H]acetyl-CoA)ImmunoassayFirefly luciferase (luc)Bioluminescence (Firefly luciferin-ATP)Galactokinaseβ-Galactosidase (lac Z)BioluminescenceColorimetric (ONPG)Colorimetric (X-GAL)Fluorescence (FDG)Fluorescence (MUGal)β-Glucuronidase (gusA, uidA)ChemiluminescenceColorimetric (X-GlcU)Fluorescence (MUGlcU)Growth hormoneImmunoassayInterleukin-2 (IL-2)Marine bacterial luciferase (Iux A/B)Bioluminescence (FMNH2-decanal)Neomycin phosphotransferase (neo)Ornithine transcarbamylasePhosphinothricin acetyltransferase (bar)Puromycin acetyltransferase (pac)Renilla luciferase (luc)BioluminescenceThaumatin IIThymidine kinaseXanthine-guaninephosphoribosyltransferaseVargula luciferaseNote:CSPD, disodium 3-(4-methoxy spiro[1,2-dioxetane-3,2′(5′-chloro)-tricyclo[3.3.1.1]decan]-4-yl phenyl phosphate;FDG, fluorescein digalactoside; X-GlcU, 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid;MUGal, 4-methylumbelliferyl β-D-galactopyranoside;MUGlcU, 4-methylumbelliferyl β-D-glucuronide;NET, nitro blue tetrazolium;ONPG, O-nitrophenyl β-D-galactopyranoside;PNPP, 4-nitrophenyl phosphate;X-GAL, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside.